Little is known about the bacterial Factors that contribute to pathogenesis of TB. However, during year 01-02 of AI-35266, the PI identified and characterized a recombinant protein encoded by M. tuberculosis that promoted uptake of latex microspheres coated with the protein into nonphagocytic cells. Subsequently (Year 03), this 27 kDa protein was discovered to be a truncated version of a native 45 kDa surface protein of M. tuberculosis encoded by a 1239-bp gene designated as mceA. The 45 kDa protein is referred to as mycobacterium cell entry protein or Mcep (the truncated protein is designated Mcep1). Reagents that affect cytoskeletal rearrangement (cytochalasin D, nocodazole, and onodansylcadaverine) inhibited the association with HeLa cells of microspheres coated with Mcep1. In addition, Mcep 1 inhibited Fc-receptor mediated oxidative burst in murine macrophage J774. A highly basic 22-amino region near the N-terminus of Mcep1 was found to be the cell uptake active domain. From the recently completed genome sequence of M. tuberculosis, the mceA locus was found to be part of a cluster of at least 15 open reading frames (ORF), 4 of which encoded a possible membrane-spanning lipoprotein, all located downstream of an ORF resembling the E. coli nta operon transcriptional regulator. The new proposal will have three specific aims. The first aim will continue the analysis of the structure and function of Mcep and its physiologic relevance to M. tuberculosis pathogenesis, focusing on characterizing the role of Mcep on inhibition of oxidative burst in macrophages and cytoskeletal rearrangement in nonphagocytic cells. The physiologic relevance of Mcep will be studied by the analysis of M. tuberculosis disrupted in mceA. The second aim will analyze the genes that cluster around the mceA locus. This aim will determine whether any of these genes constitutes part of a secretion system for the surface-expressed Mcep, and whether any of them regulates the expression of Mcep during and after entry into cells. The third aim will seek to determine whether there is a specific receptor on mammalian cells for Mcep, and, if so, its function in relation to Mcep- mediated pathogenesis. It is hoped that these studies will determine whether Mcep constitutes a virulence factor of M. tuberculosis. At the same time, they may reveal new insight into how a possible effector molecule in mycobacteria is delivered to the surface through plasma membrane and the lipid-rich cell wall layer. In addition, these studies may identify several potential targets for new chemotherapeutic agents or vaccine candidates.